E2F transcription factor-1 modulates expression of glutamine metabolic genes in mouse embryonic fibroblasts and uterine sarcoma cells.

Metabolic reprogramming is considered as a hallmark of cancer and is clinically exploited as a novel target for therapy. The E2F transcription factor-1 (E2F1) regulates various cellular processes, including proliferative and metabolic pathways, and acts, depending on the cellular and molecular context, as an oncogene or tumor suppressor. The latter is evident by the observation that E2f1-knockout mice develop spontaneous tumors, including uterine sarcomas. This dual role warrants a detailed investigation of how E2F1 loss impacts metabolic pathways related to cancer progression. Our data indicate that E2F1 binds to the promoter of several glutamine metabolism-related genes. Interestingly, the expression of genes in the glutamine metabolic pathway were increased in mouse embryonic fibroblasts (MEFs) lacking E2F1. In addition, we confirm that E2f1-/- MEFs are more efficient in metabolizing glutamine and producing glutamine-derived precursors for proliferation. Mechanistically, we observe a co-occupancy of E2F1 and MYC on glutamine metabolic promoters, increased MYC binding after E2F1 depletion and that silencing of MYC decreased the expression of glutamine-related genes in E2f1-/- MEFs. Analyses of transcriptomic profiles in 29 different human cancers identified uterine sarcoma that showed a negative correlation between E2F1 and glutamine metabolic genes. CRISPR/Cas9 knockout of E2F1 in the uterine sarcoma cell line SK-UT-1 confirmed elevated glutamine metabolic gene expression, increased proliferation and increased MYC binding to glutamine-related promoters upon E2F1 loss. Together, our data suggest a crucial role of E2F1 in energy metabolism and metabolic adaptation in uterine sarcoma cells.

Atrial Natriuretic Peptide Orchestrates a Coordinated Physiological Response to Fuel Non-shivering Thermogenesis.

Atrial natriuretic peptide (ANP) is a cardiac hormone controlling blood volume and pressure in mammals. It is still unclear whether ANP controls cold-induced thermogenesis in vivo. Here, we show that acute cold exposure induces cardiac ANP secretion in mice and humans. Genetic inactivation of ANP promotes cold intolerance and suppresses half of cold-induced brown adipose tissue (BAT) activation in mice. While white adipocytes are resistant to ANP-mediated lipolysis at thermoneutral temperature in mice, cold exposure renders white adipocytes fully responsive to ANP to activate lipolysis and a thermogenic program, a physiological response that is dramatically suppressed in ANP null mice. ANP deficiency also blunts liver triglycerides and glycogen metabolism, thus impairing fuel availability for BAT thermogenesis. ANP directly increases mitochondrial uncoupling and thermogenic gene expression in human white and brown adipocytes. Together, these results indicate that ANP is a major physiological trigger of BAT thermogenesis upon cold exposure in mammals.

Hypothalamic CDK4 regulates thermogenesis by modulating sympathetic innervation of adipose tissues.

This study investigated the role of CDK4 in the oxidative metabolism of brown adipose tissue (BAT). BAT from Cdk4-/- mice exhibited fewer lipids and increased mitochondrial volume and expression of canonical thermogenic genes, rendering these mice more resistant to cold exposure. Interestingly, these effects were not BAT cell-autonomous but rather driven by increased sympathetic innervation. In particular, the ventromedial hypothalamus (VMH) is known to modulate BAT activation via the sympathetic nervous system. We thus examined the effects of VMH neuron-specific Cdk4 deletion. These mice display increased sympathetic innervation and enhanced cold tolerance, similar to Cdk4-/- mice, in addition to browning of scWAT. Overall, we provide evidence showing that CDK4 modulates thermogenesis by regulating sympathetic innervation of adipose tissue depots through hypothalamic nuclei, including the VMH. This demonstrates that CDK4 not only negatively regulates oxidative pathways, but also modulates the central regulation of metabolism through its action in the brain.

Hepatocyte-specific deletion of Pparα promotes NAFLD in the context of obesity.

Peroxisome proliferator activated receptor α (PPARα) acts as a fatty acid sensor to orchestrate the transcription of genes coding for rate-limiting enzymes required for lipid oxidation in hepatocytes. Mice only lacking Pparα in hepatocytes spontaneously develop steatosis without obesity in aging. Steatosis can develop into non alcoholic steatohepatitis (NASH), which may progress to irreversible damage, such as fibrosis and hepatocarcinoma. While NASH appears as a major public health concern worldwide, it remains an unmet medical need. In the current study, we investigated the role of hepatocyte PPARα in a preclinical model of steatosis. For this, we used High Fat Diet (HFD) feeding as a model of obesity in C57BL/6 J male Wild-Type mice (WT), in whole-body Pparα- deficient mice (Pparα-/-) and in mice lacking Pparα only in hepatocytes (Pparαhep-/-). We provide evidence that Pparα deletion in hepatocytes promotes NAFLD and liver inflammation in mice fed a HFD. This enhanced NAFLD susceptibility occurs without development of glucose intolerance. Moreover, our data reveal that non-hepatocytic PPARα activity predominantly contributes to the metabolic response to HFD. Taken together, our data support hepatocyte PPARα as being essential to the prevention of NAFLD and that extra-hepatocyte PPARα activity contributes to whole-body lipid homeostasis.

CDK4 Regulates Lysosomal Function and mTORC1 Activation to Promote Cancer Cell Survival.

Cyclin-dependent kinase 4 (CDK4) is well-known for its role in regulating the cell cycle, however, its role in cancer metabolism, especially mTOR signaling, is undefined. In this study, we established a connection between CDK4 and lysosomes, an emerging metabolic organelle crucial for mTORC1 activation. On the one hand, CDK4 phosphorylated the tumor suppressor folliculin (FLCN), regulating mTORC1 recruitment to the lysosomal surface in response to amino acids. On the other hand, CDK4 directly regulated lysosomal function and was essential for lysosomal degradation, ultimately regulating mTORC1 activity. Pharmacologic inhibition or genetic inactivation of CDK4, other than retaining FLCN at the lysosomal surface, led to the accumulation of undigested material inside lysosomes, which impaired the autophagic flux and induced cancer cell senescence in vitro and in xenograft models. Importantly, the use of CDK4 inhibitors in therapy is known to cause senescence but not cell death. To overcome this phenomenon and based on our findings, we increased the autophagic flux in cancer cells by using an AMPK activator in combination with a CDK4 inhibitor. The cotreatment induced autophagy (AMPK activation) and impaired lysosomal function (CDK4 inhibition), resulting in cell death and tumor regression. Altogether, we uncovered a previously unknown role for CDK4 in lysosomal biology and propose a novel therapeutic strategy to target cancer cells. SIGNIFICANCE: These findings uncover a novel function of CDK4 in lysosomal biology, which promotes cancer progression by activating mTORC1; targeting this function offers a new therapeutic strategy for cancer treatment.

Interaction between hormone-sensitive lipase and ChREBP in fat cells controls insulin sensitivity.

Impaired adipose tissue insulin signalling is a critical feature of insulin resistance. Here we identify a pathway linking the lipolytic enzyme hormone-sensitive lipase (HSL) to insulin action via the glucose-responsive transcription factor ChREBP and its target, the fatty acid elongase ELOVL6. Genetic inhibition of HSL in human adipocytes and mouse adipose tissue results in enhanced insulin sensitivity and induction of ELOVL6. ELOVL6 promotes an increase in phospholipid oleic acid, which modifies plasma membrane fluidity and enhances insulin signalling. HSL deficiency-mediated effects are suppressed by gene silencing of ChREBP and ELOVL6. Mechanistically, physical interaction between HSL, independent of lipase activity, and the isoform activated by glucose metabolism ChREBPα impairs ChREBPα translocation into the nucleus and induction of ChREBPß, the isoform with high transcriptional activity that is strongly associated with whole-body insulin sensitivity. Targeting the HSL-ChREBP interaction may allow therapeutic strategies for the restoration of insulin sensitivity.

Caloric Restriction and Diet-Induced Weight Loss Do Not Induce Browning of Human Subcutaneous White Adipose Tissue in Women and Men with Obesity.

Caloric restriction (CR) is standard lifestyle therapy in obesity management. CR-induced weight loss improves the metabolic profile of individuals with obesity. In mice, occurrence of beige fat cells in white fat depots favors a metabolically healthy phenotype, and CR promotes browning of white adipose tissue (WAT). Here, human subcutaneous abdominal WAT samples were analyzed in 289 individuals with obesity following a two-phase dietary intervention consisting of an 8 week very low calorie diet and a 6-month weight-maintenance phase. Before the intervention, we show sex differences and seasonal variation, with higher expression of brown and beige markers in women with obesity and during winter, respectively. The very low calorie diet resulted in decreased browning of subcutaneous abdominal WAT. During the whole dietary intervention, evolution of body fat and insulin resistance was independent of changes in brown and beige fat markers. These data suggest that diet-induced effects on body fat and insulin resistance are independent of subcutaneous abdominal WAT browning in people with obesity.

Natriuretic peptides promote glucose uptake in a cGMP-dependent manner in human adipocytes.

Robust associations between low plasma level of natriuretic peptides (NP) and increased risk of type 2 diabetes (T2D) have been recently reported in humans. Adipose tissue (AT) is a known target of NP. However it is unknown whether NP signalling in human AT relates to insulin sensitivity and modulates glucose metabolism. We here show in two European cohorts that the NP receptor guanylyl cyclase-A (GC-A) expression in subcutaneous AT was down-regulated as a function of obesity grade while adipose NP clearance receptor (NPRC) was up-regulated. Adipose GC-A mRNA level was down-regulated in prediabetes and T2D, and negatively correlated with HOMA-IR and fasting blood glucose. We show for the first time that NP promote glucose uptake in a dose-dependent manner. This effect is reduced in adipocytes of obese individuals. NP activate mammalian target of rapamycin complex 1/2 (mTORC1/2) and Akt signalling. These effects were totally abrogated by inhibition of cGMP-dependent protein kinase and mTORC1/2 by rapamycin. We further show that NP treatment favoured glucose oxidation and de novo lipogenesis independently of significant gene regulation. Collectively, our data support a role for NP in blood glucose control and insulin sensitivity by increasing glucose uptake in human adipocytes. This effect is partly blunted in obesity.

Mitochondrial fission is associated with UCP1 activity in human brite/beige adipocytes.

OBJECTIVE: Thermogenic adipocytes (i.e. brown or brite/beige adipocytes) are able to burn large amounts of lipids and carbohydrates as a result of highly active mitochondria and enhanced uncoupled respiration, due to UCP1 activity. Although mitochondria are the key organelles for this thermogenic function, limited human data are available. METHODS/RESULTS: We characterized changes in the mitochondrial function of human brite adipocytes, using hMADS cells as a model of white- to brite-adipocyte conversion. We found that profound molecular modifications were associated with morphological changes in mitochondria. The fission process was partly driven by the DRP1 protein, which also promoted mitochondrial uncoupling. CONCLUSION: Our data demonstrate that white-to-brite conversion of human adipocytes relies on molecular, morphological and functional changes in mitochondria, which enable brite/beige cells to carry out thermogenesis.

Reactive oxygen species enhance mitochondrial function, insulin sensitivity and glucose uptake in skeletal muscle of senescence accelerated prone mice SAMP8.

Whereas reactive oxygen species (ROS) can have opposite impacts on insulin signaling, they have mainly been associated with mitochondrial dysfunction in skeletal muscle. We analyzed the relationship between these three features in skeletal muscle of senescence accelerated mice (SAM) prone (P8), which are characterized by enhanced oxidative stress compared to SAM resistant (R1). Oxidative stress, ROS production, antioxidant system, mitochondrial content and functioning, as well as in vitro and in vivo insulin signaling were investigated in gastrocnemius and quadriceps muscles. In SAMP8 compared to SAMR1, muscle content in carbonylated proteins was two-fold (p < 0.01) and ROS production by xanthine oxidase 70% (p < 0.05) higher. Furthermore, insulin-induced Akt phosphorylation measured in vivo and ex vivo as well as muscle glucose uptake measured ex vivo were significantly higher (p < 0.05). Mitochondrial respiration evidenced uncoupling and higher respiration rates with substrates of complexes II and IV, in agreement with higher maximal activity of complexes II and IV (+ 18% and 62%, respectively, p < 0.05). By contrast, maximal activity of complex I was 22% lower (p < 0.05). All strain differences were corrected after 6 months of N-acetylcysteine (NAC) treatment, thus supporting the involvement of high ROS production in these differences. In conclusion in muscle of SAMP8 compared to SAMR1, high ROS production is associated to higher insulin sensitivity and glucose uptake but to lower mitochondrial complex I activity. These conflicting adaptations, with regards to the resulting imbalance between NADH production and use, were associated with intrinsic adjustments in the mitochondrial respiration chain (mitochondrial uncoupling, enhanced complexes II and IV activity). We propose that these bioenergetics adaptations may help at preserving muscle metabolic flexibility of SAMP8.

Control of adipogenesis by oxylipins, GPCRs and PPARs.

Oxylipins are bioactive metabolites derived from the oxygenation of ω3 and ω6 polyunsaturated fatty acids, triggered essentially by cyclooxygenase and lipoxygenase activities. Oxylipins are involved in the development and function of adipose tissue and their productions are strictly related to diet quality and quantity. Oxylipins signal via cell surface membrane (G Protein-coupled receptors) and nuclear receptors (peroxisome proliferator-activated receptors), two pathways playing a pivotal role in adipocyte biology. In this review, we made an attempt to cover the available knowledge about synthesis and molecular function of oxylipins known to modulate adipogenesis, adipocyte function and phenotype conversion, with a focus on their interaction with peroxisome proliferator-activated nuclear receptor family.

miR-125b affects mitochondrial biogenesis and impairs brite adipocyte formation and function.

OBJECTIVE: In rodents and humans, besides brown adipose tissue (BAT), islands of thermogenic adipocytes, termed "brite" (brown-in-white) or beige adipocytes, emerge within white adipose tissue (WAT) after cold exposure or ß3-adrenoceptor stimulation, which may protect from obesity and associated diseases. microRNAs are novel modulators of adipose tissue development and function. The purpose of this work was to characterize the role of microRNAs in the control of brite adipocyte formation. METHODS/RESULTS: Using human multipotent adipose derived stem cells, we identified miR-125b-5p as downregulated upon brite adipocyte formation. In humans and rodents, miR-125b-5p expression was lower in BAT than in WAT. In vitro, overexpression and knockdown of miR-125b-5p decreased and increased mitochondrial biogenesis, respectively. In vivo, miR-125b-5p levels were downregulated in subcutaneous WAT and interscapular BAT upon ß3-adrenergic receptor stimulation. Injections of an miR-125b-5p mimic and LNA inhibitor directly into WAT inhibited and increased ß3-adrenoceptor-mediated induction of UCP1, respectively, and mitochondrial brite adipocyte marker expression and mitochondriogenesis. CONCLUSION: Collectively, our results demonstrate that miR-125b-5p plays an important role in the repression of brite adipocyte function by modulating oxygen consumption and mitochondrial gene expression.

Liver PPARα is crucial for whole-body fatty acid homeostasis and is protective against NAFLD.

OBJECTIVE: Peroxisome proliferator-activated receptor α (PPARα) is a nuclear receptor expressed in tissues with high oxidative activity that plays a central role in metabolism. In this work, we investigated the effect of hepatocyte PPARα on non-alcoholic fatty liver disease (NAFLD). DESIGN: We constructed a novel hepatocyte-specific PPARα knockout (Pparα(hep-/-)) mouse model. Using this novel model, we performed transcriptomic analysis following fenofibrate treatment. Next, we investigated which physiological challenges impact on PPARα. Moreover, we measured the contribution of hepatocytic PPARα activity to whole-body metabolism and fibroblast growth factor 21 production during fasting. Finally, we determined the influence of hepatocyte-specific PPARα deficiency in different models of steatosis and during ageing. RESULTS: Hepatocyte PPARα deletion impaired fatty acid catabolism, resulting in hepatic lipid accumulation during fasting and in two preclinical models of steatosis. Fasting mice showed acute PPARα-dependent hepatocyte activity during early night, with correspondingly increased circulating free fatty acids, which could be further stimulated by adipocyte lipolysis. Fasting led to mild hypoglycaemia and hypothermia in Pparα(hep-/-) mice when compared with Pparα(-/-) mice implying a role of PPARα activity in non-hepatic tissues. In agreement with this observation, Pparα(-/-) mice became overweight during ageing while Pparα(hep-/-) remained lean. However, like Pparα(-/-) mice, Pparα(hep-/-) fed a standard diet developed hepatic steatosis in ageing. CONCLUSIONS: Altogether, these findings underscore the potential of hepatocyte PPARα as a drug target for NAFLD.

In vitro brown and "brite"/"beige" adipogenesis: human cellular models and molecular aspects.

Brown adipose tissue (BAT) has long been thought to be absent or very scarce in human adults so that its contribution to energy expenditure was not considered as relevant. The recent discovery of thermogenic BAT in human adults opened the field for innovative strategies to combat overweight/obesity and associated diseases. This energy-dissipating function of BAT is responsible for adaptive thermogenesis in response to cold stimulation. In this context, adipocytes can be converted, within white adipose tissue (WAT), into multilocular adipocytes expressing UCP1, a mitochondrial protein that plays a key role in heat production by uncoupling the activity of the respiratory chain from ATP synthesis. These adipocytes have been named "brite" or "beige" adipocytes. Whereas BAT has been studied for a long time in murine models both in vivo and in vitro, there is now a strong demand for human cellular models to validate and/or identify critical factors involved in the induction of a thermogenic program within adipocytes. In this review we will discuss the different human cellular models described in the literature and what is known regarding the regulation of their differentiation and/or activation process. In addition, the role of microRNAs as novel regulators of brown/"brite" adipocyte differentiation and conversion will be depicted. Finally, investigation of both the conversion and the metabolism of white-to-brown converted adipocytes is required for the development of therapeutic strategies targeting overweight/obesity and associated diseases. This article is part of a Special Issue entitled Brown and White Fat: From Signaling to Disease.

Borage and fish oils lifelong supplementation decreases inflammation and improves bone health in a murine model of senile osteoporosis.

Fats are prevalent in western diets; they have known deleterious effects on muscle insulin resistance and may contribute to bone loss. However, relationships between fatty acids and locomotor system dysfunctions in elderly population remain controversial. The aim of this study was to analyze the impact of fatty acid quality on the age related evolution of the locomotor system and to understand which aging mechanisms are involved. In order to analyze age related complications, the SAMP8 mouse strain was chosen as a progeria model as compared to the SAMR1 control strain. Then, two months old mice were divided in different groups and subjected to the following diets : (1) standard "growth" diet - (2) "sunflower" diet (high ω6/ω3 ratio) - (3) "borage" diet (high γ-linolenic acid) - (4) "fish" diet (high in long chain ω3). Mice were fed ad libitum through the whole protocol. At 12 months old, the mice were sacrificed and tissues were harvested for bone studies, fat and muscle mass measures, inflammation parameters and bone cell marker expression. We demonstrated for the first time that borage and fish diets restored inflammation and bone parameters using an original model of senile osteoporosis that mimics clinical features of aging in humans. Therefore, our study strongly encourages nutritional approaches as relevant and promising strategies for preventing aged-related locomotor dysfunctions.

Muscle mitochondrial oxidative phosphorylation activity, but not content, is altered with abdominal obesity in sedentary men: synergism with changes in insulin sensitivity.

CONTEXT: Abdominal obesity is a major risk factor for muscle insulin resistance. Mitochondria may play a key role in this etiology. OBJECTIVE: Changes in muscle mitochondrial content and function were examined according to abdominal obesity and insulin sensitivity in men. STUDY DESIGN AND SETTING: The descriptive MitHyCal study was conducted on the general population of Clermont-Ferrand, France. PARTICIPANTS: Forty-two healthy sedentary men (41.7 +/- 4.3 yr) were divided into four groups according to waist circumference: 87 cm or less (group 1, n = 10); 88-93 cm (group 2, n = 12); 94-101 cm (group 3, n = 10); and 102 cm or greater (group 4, n = 10). INTERVENTION: Plasma metabolic check-up was performed, and insulin sensitivity index was calculated from glucose and insulin responses to a 3-h oral glucose tolerance test. Muscle biopsies were obtained to assess mitochondrial content, oxidative phosphorylation activity, and superoxide anion (reactive oxygen species) production. MAIN OUTCOME MEASURES: Assessment of muscle mitochondrial content and function was planned before data collection began. RESULTS: Abdominal obesity was negatively correlated to insulin sensitivity index (r = -0.39; P < 0.01), and only group 4 was insulin-resistant (P < 0.05). There were no between-group differences in muscle mitochondrial content and maximal activity of key oxidative enzymes. In contrast, muscle mitochondrial ADP-stimulated respiration rate was 24% higher in groups 2 and 3 compared to groups 1 and 4 (P < 0.05). Mitochondrial ATP and reactive oxygen species production rates were 27 and 48% lower in group 4 than in group 1 (P < 0.05). CONCLUSION: Abdominal obesity is associated with alterations in intrinsic muscle mitochondrial function but not content. These adaptations mainly result in reduced mitochondrial ATP production rate in response to insulin resistance.

Method for functional study of mitochondria in rat hypothalamus.

Different roles of mitochondria in brain function according to brain area are now clearly emerging. Unfortunately, no technique is yet described to investigate mitochondria function in specific brain area. In this article, we provide a complete description of a procedure to analyze the mitochondrial function in rat brain biopsies. Our two-step method consists in a saponin permeabilization of fresh brain tissues in combination with high-resolution respirometry to acquire the integrated respiratory rate of the biopsy. In the first part, we carefully checked the mitochondria integrity after permeabilization, defined experimental conditions to determine the respiratory control ratio (RCR), and tested the reproducibility of this technique. In the second part, we applied our method to test its sensitivity. As a result, this method was sensitive enough to reveal region specificity of mitochondrial respiration within the brain. Moreover, we detected physiopathological modulation of the mitochondrial function in the hypothalamus. Thus this new technique that takes all cell types into account, and does not discard or select any mitochondria sub-population is very suitable to analyze the integrated mitochondrial respiration of brain biopsies.